Total proteins were isolated and pooled for use in Western blots as previously described.16 (link) Briefly, fresh segments of MAs were frozen in liquid nitrogen and stored at −80°C until assay. After pulverization, frozen MAs were homogenized in ice cold Tris-EDTA buffer. Protein concentration was determined using a protein assay (Bio-Rad Laboratories; Hercules, USA). Equivalent amounts (60 μg) of total protein from the MAs were added to the adjacent lanes. The related primary antibody was mouse monoclonal antibodies for eNOS (1:1000, Transduction Laboratories, Lexington, UK). The secondary antibody was anti-mouse IgG-HRP (1:8000, Proteintech Group Inc., Chicago, Illinois, USA). The immunoreactive bands were visualized with enhanced chemiluminescence, and the signals were recorded with Bio-Rad ChemiDOC XRS+ (Bio-Rad Laboratories, Hercules, CA, USA). GAPDH was detected and used to correct for the equal loading of all of the samples. The eNOS protein content was expressed relative to the GAPDH content.
Anti mouse igg hrp
Manufactured by Proteintech
Sourced in United States
Anti-mouse IgG-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP) that can be used to detect and quantify mouse immunoglobulin G (IgG) in various immunoassays. It provides a convenient and sensitive means of amplifying the signal for mouse IgG detection.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit igg hrp, by Proteintech (2 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Kdm3a, by Abcam (1 mentions)
Ripa lysate, by Beyotime (1 mentions)
Gapdh, by Proteintech (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Gapdh 60004 1 lg, by Proteintech (1 mentions)
Foxk1, by Cell Signaling Technology (1 mentions)
Ecl chemiluminescence kit, by Merck Group (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Anti rabbit igg hrp, by Cell Signaling Technology (1 mentions)
Cyclin e1, by Cell Signaling Technology (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
Anti piezo1, by Proteintech (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
4 protocols using anti mouse igg hrp
1
Western Blot Analysis of eNOS Protein in Mesenteric Arteries
2
Western Blot Quantification of Protein Targets
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Western blot assays were performed using the methodology as previously described.26 (link)
Cells were lysed with RIPA lysate (Beyotime Biotechnology), and cell lysates were prepared. Proteins were separated by 10% SDS-PAGE. Proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes. Antibodies used in this study were as follows: KDM3A (diluted 1:1000; Cat# ab191389; Abcam), ENO3 (diluted 1:1000; Cat# ab119327; Abcam), GAPDH (diluted 1:5000; Cat No.60004-1; Proteintech), anti-Rabbit-IgG-HRP (diluted 1:10000; Cat No.PR30012; Proteintech), and anti-Mouse-IgG-HRP (diluted 1:10000; Cat No.PR30012; Proteintech). ECL reagent was applied to detect the signals.
Cells were lysed with RIPA lysate (Beyotime Biotechnology), and cell lysates were prepared. Proteins were separated by 10% SDS-PAGE. Proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes. Antibodies used in this study were as follows: KDM3A (diluted 1:1000; Cat# ab191389; Abcam), ENO3 (diluted 1:1000; Cat# ab119327; Abcam), GAPDH (diluted 1:5000; Cat No.60004-1; Proteintech), anti-Rabbit-IgG-HRP (diluted 1:10000; Cat No.PR30012; Proteintech), and anti-Mouse-IgG-HRP (diluted 1:10000; Cat No.PR30012; Proteintech). ECL reagent was applied to detect the signals.
3
Western Blot Analysis of Protein Expression
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The proteins of tissues and cells were lysed in M-PER Protein Extraction Reagent (Pierce) mixed with protease inhibitor cocktail. BCA assay kit (Pierce) was used to determine the protein concentrations. The proteins were then separated on 8% SDS-PAGE, and transferred onto nitrocellulose membranes. After blocked with 5% non-fat milk for 1.5 h at room temperature, the membrane were incubated with primary antibodies overnight at 4°C, and followed by incubation with the corresponding secondary antibodies for 1.5 h. The blots were detected by ECL chemiluminescence kit (Millipore). These primary antibodies were obtained from Cell Signaling Technology (Danvers, USA), FOXK1 (# 12025), mTOR (# 2983), p-mTOR (# 5536), AKT (# 4691), p-AKT (# 4060), Cyclin D1(# 2922), Cyclin E1 (# 4129), E-cadherin (# 3195), N- cadherin (# 13116), CDK4 (# 12790), CDK6 (# 3136), and anti-Rabbit IgG-HRP (# 7074). The antibody against vimentin (#ab8979) was purchased from Abcam (Cambridge, USA). The antibody against Gapdh (# 60004-1-lg) and anti-Mouse IgG-HRP (# SA00001-1) were purchased from Proteintech (Rosemont, USA). Selected blots were quantified by using Image J (NIH, USA).
4
Immunoblotting of PIEZO1 Membrane Protein
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Membrane protein samples were isolated and boiled in protein loading buffer for 10 min. For PIEZO1, 8% SDS PAGE was performed and protein was transferred to nitrocellulose membrane at 250 mA for 6 h. Anti‐PIEZO1 (1:500, Proteintech, 15,939‐1‐AP) was applied in TBST 5% milk overnight at 4°C and anti‐Rabbit IgG‐HRP (1:5000, Proteintech, SA00001‐2) was incubated for 2.5 h at room temperature. ACTB antibody (1:5000, Proteintech, 66,009‐1‐Ig) was used as internal control and anti‐mouse IgG‐HRP (1:5000, Proteintech, SA00001‐1) was applied as recommended. Other protein immunoblotting was conducted accordingly. Bands were detected and visualized with ECL reagent (Thermo Fisher, 32,109). In consideration of the huge gap of stripe strength between PIEZO1 and the internal control, the membrane was cut and developed, respectively.
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